This proposal integrates multiple aspects of molecular virology, host-pathogen interactions, and neuronal biology and highlights the skills and expertise that Dr. Matthew P. Taylor has developed during a career of scientific investigation. The experiments detailed in the research strategy extend upon Dr. Taylor's research findings and expertise in neuronal culture, live cell imaging, and DNA and RNA virology. His 17 years of laboratory experience in both academic and industrial settings and a broad background in molecular biology techniques allow him to develop a wide range of experimental models. This experience was refined during Dr. Taylor's graduate and post-doctoral research into the viral replication and spread of both poliovirus and herpesvirus. His 20 publications, including 2 seminal reviews and 4 first author publications in virus-cell interaction, are demonstrative of significant productivity and impact in the field of virology. The research strategy builds off of the recent publication, Alphaherpesvirus axon-to-cell spread involves limited virion transmission in PNAS, which describes the novel observation that directional spread of Herpes Simplex Virus (HSV) and Pseudorabies virus (PRV) involves the transmission of a single viral particle to initiate infection. This population bottleneck has important implications on viral infection and human health, as directional spread is fundamental to the formation of recurrent lesions associated with HSV infection. The research proposed in this application builds off the methodologies of axon-to-cell spread quantification to understand the nature and mechanism of the population bottleneck. [Aim 1 seeks to identify important viral proteins involved in regulating axon-to-cell spread of infection. Previously characterized viral mutants and novel mutants isolated from a random mutagenic screen will be combined with published assays to quantify anterograde spread. By understanding which proteins are involved in regulating virion transmission, we may understand the mechanism behind the population bottleneck. Aim 2, investigates the role of cellular antiviral signaling and activation of interfern responses in restricting the number of virions that initiate infection in susceptible cell populations. Cellular antiviral signaling can be activated during HSV entry upon cell-free inoculation, but has not been characterized for axon-to-cell transmission of limited numbers of virions. A potential role of antiviral signaling will be elucidated through protein localization, fluorescent reporter cell lines, and deletion mutants of both cellular and viral proteins.] Aim 3 wll determine the impact on spread of the axon-to-cell population bottleneck of herpesviruses in an infected host. A mixture of fluorescent protein expressing HSV and PRV recombinants will be injected into the eye to following the diversity of infection. The three-color fluorescent protein expressing mixture of viruses provides a visual read-out of viral genome expression, allowing the visualization and quantitation of co-infection at the discrete sites of primary infection in th eye and at well-described sites of axon-to-cell spread in the brain. Determining the presence of a spread bottleneck in vivo will allow the testing of results from the prior aims on the spread of viral infection within an infected host. The experiments in this proposal utilize a number of Dr. Taylor's experimental strengths and expand into new areas of experimental methodology. To assure the highest level of success, he will continue to gain experience in neurobiology and imaging through coursework and collaboration at his future institution. He has received training in animal models of infection, dissection and histology is from a long-time collaborator of the Enquist lab, Dr. J. Patrick Card a the University of Pittsburgh. Dr. Card is an expert in animal models of PRV and HSV infection, having performed the first work describing the high fidelity neural circuit tracing capacity of PRV in the rodent eye. The combination of training and experimentation will provide a solid foundation in support of Dr. Taylor's goal to be an independent researcher. The work described here will be vital to the initiation of his research pro- gram and provide important preliminary results in support of furthe grant applications. Answering the fundamental questions that underlie axon-to-cell transmission of herpesviruses will open up new lines of questioning regarding the viral interactions with the axonal signaling, neuro-immune regulation, and the general regulation of neurotropic viral infection in an infected host. Dr. Taylor is excited to share his training and expertise with the next generation of scientists while answering fundamental questions of viral infection.